So-called endotoxins (intracellular toxins) mainly include fragments of cells of bacteria that are not stained by Gram staining (Gram-negative), and a component of the fragments is a lipid-polysaccharide called a lipopolysaccharide. To be specific, the component is a lipopolysaccharide (LPS) in which a lipid called Lipid A and a polysaccharide chain are bonded via 2-keto-3-deoxyoctonate (KDO). A lipid component called Lipid A included in the lipopolysaccharide is bonded to a cellular receptor, causing inflammation, and causing a variety of severe clinical symptoms in many cases. The endotoxins are, as described above, substances causing clinical symptoms such as sepsis and bacteremia that are high in fatality. Thus, estimation of the endotoxins present in the body is highly demanded clinically.
Further, it is important that medicinal products (such as injections) and medical devices (such as angiocatheters) are free of endotoxin contamination (pyrogen-free), and it is strictly required that endotoxins be completely removed from medicinal products (such as recombinant proteins and DNA used for gene therapies) prepared by using bacteria.
In confirmation of the removal of endotoxins or measurement of endotoxins in emergency medicine, promptness is required for attaining the purposes of copying with a large number of measuring samples and carrying out life-saving treatment.
Research has been made since old days on measuring the value of endotoxins for the treatment of sepsis or the like. Since the discovery of a fact that a factor group contained in the component of an amebocyte of a horseshoe crab (Limulus polyphemus) specifically reacts with endotoxins, resulting in gelation, trials for quantifying the endotoxins have been made by using limulus amebocyte lysates (LAL reagent or limulus reagent).
A measuring method in which the limulus reagent was used for the first time was a simple measuring method called a gelation method, in which plasma from a patient serving as a sample is mixed with the limulus reagent, the mixture is left to stand still, the mixture is positioned up side down after a certain time, the presence or absence of gelation is confirmed by whether or not the solution is solidified, and the amount of endotoxins is estimated based on the maximum dilution ratio at which the gelation is caused.
Later, attention has been paid to the increase of turbidity during a gelation reaction. As a result, there is known a turbidimetric assay, in which a turbidimeter using an optical measuring method is used to measure an endotoxin concentration based on changing speed in turbidity involved in the gelation reaction.
In addition, a synthetic chromogenic substrate method has already been known, in which method a gelation reaction causing a conversion from coagulogen to coagulin is replaced by a chromogenic reaction of a synthetic substrate in the final stage of a reaction process caused by a limulus reagent. This is a method in which a synthetic chromogenic substrate (Boc-Leu-Gly-Arg-p-nitroanilide) is added in place of a coagulation precursor (coagulogen) in a coagulation process, the hydrolysis of the synthetic chromogenic substrate then produces free p-nitroaniline, and the colorimetric analysis of the resultant yellow chromogenic development is performed to measure an endotoxin concentration.
Besides, the following measuring apparatuses disclosed in Patent Documents 1 and 2 are exemplified as a conventional gelation reaction measuring apparatus or a measuring apparatus associated with the conventional gelation reaction measuring apparatus.
Patent Document 1 does not relate to a gelation reaction measuring apparatus, but relates to a method of measuring the size and number of the aggregated clumps of platelets in blood at each of the processes in which the platelets aggregate and grow as clumps. This is a method in which a sample in a sample cuvet is irradiated with an illuminating radiation from a laser light source, the scattered light that has been scattered laterally by 90° because of the presence of the platelets is partially detected with a photodetector, and the size and number of the aggregated clumps of the platelets are measured based on the detection results.
Further, Patent Document 2 relates to a gelation reaction measuring apparatus using a turbidimetric assay. This is an assay in which the time-dependent changes of the intensity of transmitted light in a mixture obtained by mixing a specimen (sample) and a limulus reagent are measured, and an endotoxin concentration in the specimen is measured based on the amounts of the changes in a predetermined time.
Further, Patent Document 3 relates to a gelation reaction measuring apparatus for measuring the concentration of a target substance such as endotoxins in a sample via a gelation reaction. This includes a photoreceptive element for receiving a laser beam of light scattered by gel particles generated in a sample cuvet, and measuring means for measuring the diameter of the gel particles and the number thereof on time series based on the output of the scattered light detection of the photoreceptive element.
According to Patent Documents 1-3, when the sample in the sample cuvet is irradiated with the illuminating radiation from the laser light source, the scattered light which leaves for the side that is different from the side installed the laser light source; for example the front scattered light which leaves for the front side which is opposite the laser light source or the other side scattered light which leaves for the other side laterally by 90° from the laser light source is detected with the photodetector, and these gelation reaction measuring apparatus measure the aggregated clumps of the platelet which is measurement subject or the endotoxin concentration based on detected result of the photodetector.
Moreover, measuring techniques using a gelation reaction are used for measuring not only the endotoxins described above but also β-D-glucans or the like.
β-D-glucans are polysaccharides constituting cell membranes specific to fungi. Measurement of the β-D-glucans is effective for screening a wide variety of fungi responsible for fungal infection, including not only fungi found in a general clinical environment, such as Candida, Aspergillus, or Cryptococcus, but also fungi rarely found in the general clinical environment.
The phenomenon in which a component extracted from a amebocyte of a limulus is gelated with β-D-glucans is also used in the measurement of β-D-glucans, and the above-mentioned gelation method, turbidimetric assay, or synthetic chromogenic substrate method is used to carry out the measurement.
Measuring techniques of endotoxins and of β-D-glucans have common points. For example, almost the same kind of measuring hardware is used to remove a Factor G component from the components extracted from amebocyte of Limulus, and hence a gelation reaction or chromogenic reaction selective to endotoxins can be measured. Alternatively, endotoxins in a sample are inactivated by pretreatment, and hence a gelation reaction or chromogenic reaction selective to β-D-glucans can be measured.
Patent Document 1: JP 3199850 B2 (Examples and FIG. 1)
Patent Document 2: JP 2004-93536 A (Modes for carrying out the Invention and FIG. 3)    Patent Document 3: WO2008/038329 A (Best mode for carrying out the invention and FIG. 1)